蚓激酶生物活性检测方法的研究

本文采用组织纤溶酶原激活剂（ＴＰＡ）─纤维蛋白平板法测定蚓激酶的生物活性。该法操作简便，易于观察，重现性好。     

TPA诱导的血小板聚集的研究

应用不同浓度ＴＰＡ诱发血小板聚集，观察２０名正常人血小板的聚集率。结果显示：ＴＰＡ从５ｎｇ／ｍｌ到１０ｎｇ／ｍｌ之间，随着浓度的增加，血小板聚集率逐渐增加，最佳浓度为１０ｎｇ／ｍｌ。当ＴＰＡ浓度〉１０ｍｇ／ｍｌ时，继续增加ＴＰＡ浓度，血小板聚集率将不再升高，且于２０ｍｇ／ｍｌ时出现明显的解聚现象。     

CLOTBUST: Design of a Randomized Trial of Ultrasound-Enhanced Thrombolysis for Acute Ischemic Stroke

BACKGROUND: Intravenous tissue plasminogen activator (TPA) therapy can be monitored with 2 MHz transcranial Doppler (TCD). This article describes the design of CLOTBUST (combined lysis of thrombus in brain ischemia using transcranial ultrasound and systemic TPA), the first prospective international multicenter randomized clinical trial of noninvasive externally applied ultrasound to enhance systemic thrombolysis in human stroke. SUBJECTS: Patients with acute ischemic stroke eligible for intravenous TPA therapy within 3 hours of symptom onset who have detectable middle cerebral artery occlusion on a prebolus TCD are included in this trial. All patients receive standard 0.9 mg/kg TPA therapy. Patients are randomized (1:1) to either 2 hours of continuous monitoring with TCD or placebo monitoring. FDA-approved portable diagnostic TCD equipment and standard headframes (Marc series, Spencer Technologies, Seattle, WA) are used. Output of TCD units is set at 100% power achievable at depths of insonation that display the worst TIBI flow grade signals. METHODS AND END-POINTS: Acute MCA occlusion on prebolus TCD is defined as thrombolysis in brain ischemia (TIBI) flow grades 0-3. Treating physicians are blinded to randomization assignment, and certified scorers measure stroke severity using the National Institute of Health Stroke Scale (NIHSS). Safety of continuous TCD monitoring is determined by rates of symptomatic (NIHSS score increase by 4+ points) intracerebral hemorrhage within 72 hours after initial symptom onset. Potential enhancement of TPA therapy will be determined using combined primary end-point of early complete recanalization on TCD (TIBI flow grades 4-5), dramatic recovery (NIHSS < or = 3 points), or decline in the NIHSS > or = 10 points repeatedly measured every 30 minutes within 2 hours after TPA bolus. Other end-points include recovery at 24 hours and 3 months, modified Rankin scores (mRS) are obtained at 90 days, and favorable outcome is determined as NIHSS or mRS scores 0-1. CONCLUSIONS: The aim of phase II CLOTBUST trial is to determine the rates of early complete recanalization and dramatic/early clinical recovery in TPA + TCD and TPA groups. The sample size is set at 126 patients since a medium effect size (.50) is anticipated for TPA + TCD group vs TPA alone to achieve combined primary end-point.     

促癌变剂对淋巴细胞核仁rRNA基因转录的活化作用——rDNA原位杂交荧光显示法

用原位杂交荧光显示法观察了人淋巴细胞在促癌变剂黄芫花提取物(WCE)和12-0-十四烷巳豆醇-13乙酸酯(TPA)处理后,间期核仁rDNA的定位与数量改变,并与丝裂原植物血细胞凝集素(PHA)的效应作了比较,同时用银染色法观察了核仁。对照组淋巴细胞核仁小,原位杂交的rDNA为少数明亮荧光斑和分散的荧光点。经促癌变剂WCE和TPA处理后,银染色的核仁增大,银染颗粒增多,表明rDNA转录活化。原位杂交证明rDNA信号数目明显扩增,许多荧光小点断续相连形成网织状结构,与PHA刺激核仁转录活化的表现一致。对核仁内所含银染颗粒和rDNA荧光斑点数均值的统计学分析表明,WCE、TPA和PHA各加药组均明显多于对照组。3个加药组之间无明显差别。提示两种促癌变剂皆具有刺激核仁rDNA扩增和转录活化的效应。     

TPA-induced cohort migration of well-differentiated human rectal adenocarcinoma cells: cells move in a RGD-dependent manner on fibronectin produced by cells, and phosphorylation of E-cadherin/catenin complex is induced independently of cell-extracellular matrix interactions

 We have already presented a two-dimensional cell motility assay using a highly metastatic variant (L-10) of human rectal adenocarcinoma cell line RCM-1 as a motility model of tumour cells of epithelial origin. In this model, L-10 cells showed locomotion as a coherent sheet when stimulated with 12-O-tetradecanoylphorbol-13-acetate (TPA), and we called this type of movement ”cohort migration”. Electron and immunoelectron microscopic study of the migrating cell sheets demonstrated localized release from cell–cell adhesion only at the lower portion of the cells with loss of E-cadherin immunoreactivity, and this change was associated with increased tyrosine phosphorylation of the E-cadherin–catenin complex, including β-catenin. Cell–extracellular matrix (ECM) interactions involved in this TPA-induced cohort migration and their effect on tyrosine phosphorylation of the E-cadherin-catenin complex have now been investigated. L-10 cell cohort migration was almost completely inhibited by addition of Arg-Gly-Asp (RGD) peptide into the medium, and thus RGD dependent. Cohort migration was stimulated on type I and IV collagens, fibronectin (FN)- and laminin-coated substratum, but was inhibited by RGD only on FN-coated surface. By using immunofluorescent techniques, FN was demonstrated preferentially around migrating cells, and a protein synthesis inhibitor, cycloheximide, inhibited the migration by about 75%. FN produced by L-10 cells were found to be mostly EDA+ FN when analysed by RT-PCR. Moreover, anti-FN antibody, but not anti-vitronectin antibody, inhibited the TPA-induced cohort migration almost completely. Thus, it was likely that L-10 cells produced FN themselves and moved on the FN substrate in an RGD-dependent manner. However, stimulation of migration by type I collagen coating and inhibition by RGD treatment did not affect the tyrosine phosphorylation of the E-cadherin–catenin complex induced by TPA, indicating that cell–cell interactions were adjusted to suit cell migration, irrespective of the condition of cell–ECM adhesion, during TPA-induced cohort migration. Received: 31 December 1997 / Accepted: 2 April 1998     

颈静脉孔区的显微解剖及定位标志研究

目的:研究颈静脉孔区的显微外科解剖,探讨寰椎横突(TPA)、头侧直肌、二腹肌沟、颈静脉突等解剖标志在颈静脉孔区病变手术中的定位意义。方法:成人头颈标本15例,男13例,女2例,红色乳胶灌注颈总动脉和椎动脉。手术显微镜下(×3～×30)逐层显露颈静脉孔区结构,明确该区显微解剖特征、相关的解剖标志及其定位意义。结果:颈静脉孔区多数重要的解剖结构均可以TPA为参照标志予以明确。二腹肌后腹位于其浅层。TPA的后方为枕下三角,三角内有椎动脉、椎静脉丛和颈1神经通过。头侧直肌起始于TPA的外表面,止于枕骨颈静脉突的下表面,可作为确定颅外颈静脉孔的解剖标志。茎突位于TPA的前方,颈内静脉、迷走神经、副神经、舌下神经穿行于茎突与TPA之间,颈内动脉位于颈内静脉的前内侧。二腹肌、颈静脉突、颈动脉嵴对定位面神经、颈静脉孔及舌咽神经等结构具有重要意义。结论:颈静脉孔区解剖结构复杂,利用寰椎横突、头侧直肌、二腹肌沟、颈静脉突等解剖标志有助于明确此区域重要的解剖结构,避免术中不必要的损伤。     

Lineage relationship of chronic lymphocytic leukemia and hairy cell leukemia: studies with TPA

The tumor promoting agent TPA (phorbol ester; 1.6 X 10(-8)M) was used to induce the differentiation in vitro of B-chronic lymphocytic leukemia (B-CLL) cells from 14 untreated patients. The uninduced phenotype was SIg+, Mrbc+, RFT-1+, RFA-4-, FMC7-. After 72 h incubation with TPA, B-CLL cells became RFA-4+, FMC7+ and lost the capability of Mrbc rosetting. Large proportions of the "induced" cells also showed morphological and ultrastructural changes, such as undulating membranes and bleblike protusions and became strongly positive for tartrate resistant acid phosphatase (TRAP+) and also contained cytoplasmic immunoglobulins. These features are very similar to the features of hairy cell leukemia (HCL). These observations confirm previous clinical findings that B-CLL and HCL are related disorders of the B lineage. The development of "hairy" features in induced B-CLL and in HCL seems to be a malignancy-associated feature because the Mrbc+ normal B cells (B-CLL-equivalent cells) isolated from tonsil also develop TRAP positivity but no membrane aberrations.     

Modulation of natural killing by tumor promoters: the regulatory influence of adherent cells varies with the type of target cell

We have analyzed the regulatory effects of two classes of tumor promoters, phorbol diesters and indole alkaloids, on human natural killer (NK) cell activity in vitro. In accordance with previous reports, we found that 12-O-tetradecanoylphorbol-13-acetate (TPA) inhibited natural killing against K 562 targets by unseparated mononuclear cells. Here, suppression of NK required the presence of adherent cells (macrophages). Contrary to the results obtained with K 562, tumor promoter-induced suppression of NK activity tested against U 937, another cell line of known NK susceptibility, was independent of the presence of adherent cells. Thus, NK cytotoxicity of effector cells rigorously depleted of adherent and Ia-positive cells was still inhibited when assayed against U 937, while it was generally enhanced when tested against K 562. Identical results were obtained with teleocidin and dihydroteleocidin B, two members of the recently discovered indole alkaloid class of tumor promoters. Therefore, we demonstrate that the regulatory effect of tumor promoters on human NK activity (suppression or stimulation) is determined not only by macrophages at the effector cell level but also by the type of target cell under study.     

重组组织纤溶酶原激活物预防蛛网膜下腔出血后迟发性脑血管痉挛的实验研究

目的实验研究重组组织纤溶酶原激活物预防蛛网膜下腔出血后迟发性脑血管痉挛(DVS).方法实验选取12只家犬,随机分成两组.采取"两次出血法"制成蛛网膜下腔出血(SAH)模型.SAH前先做基底动脉造影,然后行枕大池穿刺,抽出脑脊液4ml后注入等量自体动脉血.第一次"SAH"后48小时再次注入自体动脉血4ml.第二次注血后6小时治疗组6只动物经枕大池穿刺注入组织型纤维蛋白溶解酶原激活物(r-TPA)25mg;对照组注入生理盐水.7天后再次行基底动脉造影.结果动脉造影r-TPA治疗组基底动脉口径无明显变化(P＞0.05);解剖除1例基底动脉外膜上可见数点凝血外,其余动物颅底均无血块.对照组两次动脉造影基底动脉缩小极为明显(P＜0.01),有严重的血管痉挛.颅底充满血块,基底动脉被血块所包绕.结论r-TPA能充分地溶解未成熟的(SAH后48小时)蛛网膜下腔凝血块,从而有效的预防迟发性脑血管痉挛的出现.